Timothy grass antigen specific anti-idiotypic antibodies

ABSTRACT

This invention provides a number of immunological reagents useful for the suppression of allergic reactions induced by Timothy-grass pollen allergen (or cross-reacting allergens thereof) and for methods of obtaining said reagents. The reagents include: (1) an anti-idiotypic antibody (anti-IgE id ) directed to the allergen (Timothy grass antigen) binding site of an IgE immunoglobulin; (2) an anti-idiotypic antibody directed to the allergenic T-cell helper factor (T HF ) derived from Timothy grass pollen antigen stimulated lymphocytes; (3) unique methods for generating antigen-specific T-suppressor cells (T S ); (4) a unique method for producing antigen-specific suppressor T cell factors (T SF ); (5) a method for fractionation of T SF  ; and (6) a specific T cell factor, T SF2 . The invention further provides pharmaceutical compositions comprising the above anti-idiotypic antibodies or suppressor factor derived from T s  cells.

BACKGROUND OF THE INVENTION

The nature of the allergic reaction or response in humans and animals to various antigens is not completely understood. It is theorized that the allergic response to a foreign antigen, such as the antigen of grass pollen, involves the interaction of at least two different cell types. One of these is conventionally termed a T-cell, or Thymus derived lymphocyte, and the second a B-cell, which is a bone marrow derived lymphocyte. Each of these two cells, the T and the B lymphocytes, recognize different parts of the antigenic protein that causes the allergic response. The B-lymphocyte recognizes the antigenic determinant whereas the T-lymphocyte recognizes a different portion of the protein, termed a carrier determinant. The B-cells of an animal sensitive to the antigen, upon recognition of the antigenic determinant of the antigen, produce antibodies to the antigen which give rise to the allergic reaction or response. T-cells do not produce the circulating antibody that is involved in allergic responses, but regulate their production by the B-cells. One sub-population of T-cells which assists in making antibodies are the so-called helper T-cells (T_(H) cells). They effectively cooperate or interact with the B-lymphocyte to produce antibodies. Another sub-population of T-cells, the suppressor T-cell (T_(S) cells), effectively suppresses the action of helper T-cells so that they cannot participate with B-lymphocytes to produce antibodies. It is thought that the T_(S) cells also act on the B-cells themselves to inhibit the formation of antibodies.

The allergenic antibody is produced by the B-cells, as noted above, after recognition of the antigenic determinant of the antigen. This determinant is a peptide segment of the antigen protein molecule. In Timothy grass pollen the whole allergenic antigen is conventionally referred to as antigen B. All other grass antigens cross reactive with antigen B possess the same antigenic determinant as antigen B.

The peptide segment of the antibody protein molecule which binds to the antigenic determinant is conventionally referred to as the idiotype or idiotypic determinant. This same idiotypic determinant also exists in T and B cells as receptors.

Unsuccessful attempts have been made in the past to inhibit the allergic response in animals sensitive to various allergenic antigens by enhancing the T_(S) cell population in such animals or affecting the T_(H) cell population thereof.

It is an object of the present invention to provide an anti-idiotypic antibody which is directed against Timothy grass antigen B- specific (or any cross reactive grass antigen-specific) antibody.

It is a further object of the invention to provide T_(S) cells and suppressor factors derived therefrom which directly affect the ability of T_(H) -cells and B-cells to produce allergenic antibody to the grass antigens.

It is still a further object of the invention to provide a pharmaceutical composition and method for treatment of animals, including humans, sensitive to Timothy grass antigen and other grass antigens cross reactive therewith.

SUMMARY OF THE INVENTION According to the present invention there is provided a method of preparing an anti-idiotypic antibody directed against (1) Timothy grass antigen B- and (2) cross reactive grass antigen-specific allergenic antibody, IgE. The anti-idiotypic antibody, anti-IgE_(id), is specifically reactive with the idiotypic determinants on IgE and (1) antigen B- and (2) cross reactive grass antigen-specific T and B cells and is prepared by:

(1) immunizing a first animal species with a source of antigen B,

(2) collecting sera from the immunized first animal species,

(3) separating IgE from the sera,

(4) immunizing a second animal species with the IgE,

(5) collecting sera from the immunized second animal species, and

(6) separating anti-IgE_(id) antibody from the sera.

The present invention also provides a method of preparing an anti-idiotypic antibody directed against (1) Timothy grass antigen B- and (2) cross reactive grass antigen-specific allergenic T-helper factor, T_(HF). The anti-idiotypic antibody, anti-T_(HF), is specifically reactive with the idiotypic determinants on the T_(HF) and (1) antigen B- and (2) cross reactive grass antigenspecific T and B cells and is prepared by:

(1) immunizing a first animal species with photooxidized antigen B in alum,

(2) collecting T_(H) -cells from the immunized first animal species,

(3) culturing the T_(H) -cells with a source of Timothy grass antigen B or cross reactive grass antigen,

(4) separating T-helper factor T_(HF) from the culture,

(5) immunizing a second animal species with the T-helper factor, T_(HF),

(6) collecting sera from the immunized second animal species, and

(7) separating anti-T_(HF) antibody therefrom.

The invention also provides a method of producing (1) Timothy grass antigen B- and (2) cross reactive grass antigen-specific T suppressor cells, T_(S), comprising:

(1) culturing anti-IgE_(id) or anti-T_(HF) with normal cells of an animal to induce the formation of T_(S) cells in the culture, and

(2) separating the T_(S) cells from the culture.

The invention further provides a method of producing T suppressor cells, T_(S), comprising:

(1) immunizing an animal species with photooxidized antigen B, and

(2) collecting T_(S) cells from the immunized animal species.

The invention provides a further method of producing T suppressor cells, T_(S), comprising:

(1) immunizing an animal species with anti-IgE_(id) or anti-T_(HF), and

(2) collecting T_(S) from the immunized animal species.

The invention also provides a method of producing (1) Timothy grass antigen B- and (2) cross reactive grass antigen-specific suppressor T-cell factor, T_(SF), by extraction from T_(S). T_(SF) is actually a mixture of at least two other proteins, designated T_(SF).sbsb.1 and T_(SF).sbsb.2. Only T_(SF).sbsb.1 is Timothy antigen binding. T_(SF).sbsb.2, while not antigen binding, will bind to the idiotype expressed in T_(SF).sbsb.1 or T_(HF).sbsb.1 or the Ig molecule itself.

The invention provides a further method for producing T_(SF), comprising:

(1) culturing T suppressor cells, T_(S), with antigen-IgE_(id), anti-T_(HF) or antigen B, and

(2 )separating T_(SF) from the cell containing culture.

The invention provides an additional method of producing T suppressor cells, T_(S), comprising:

(1) culturing T_(SF) with normal cells of an animal species, and

(2) separating T_(S) from the normal cell containing culture.

The invention provides a method of fractionating T_(SF) into separate factors T_(SF).sbsb.1 and T_(SF).sbsb.2 comprising subjecting T_(SF) to affinity chromatography on an adsorbent comprising Timothy grass antigen D, on an inert carrier substrate which binds T_(SF).sbsb.1.

According to the present invention there is also provided a pharmaceutical composition in unit dosage form for suppressing the allergic response of an animal sensitive to Timothy grass antigen B or a cross reactive grass antigen comprising an anti-allergic response effective amount of anti-IgE_(id), anti-T_(HF), T_(SF), T_(SF).sbsb.1 or T_(SF).sbsb.2 and a pharmaceutically acceptable carrier.

The present invention also provides a method for suppressing the allergic response of an animal sensitive to Timothy grass antigen B or a cross reactive grass antigen comprising administering to the animal an anti-allergenic response effective amount of anti-Ig_(id), anti-T_(HF), T_(SF), T_(SF).sbsb.1 or T_(SF).sbsb.2.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood by those skilled in the art that the term "animal" as used herein and in the appended claims includes humans.

The present invention is predicated on the following theory of immune response by induction of T-cell suppression. As noted above the T_(H) (helper) cells interact with B-cells in a sensitive or "allergic" animal upon recognition of the carrier and antigenic determinants, respectively, of the grass antigen, to produce the allergic antibody IgE. Administration to the animal of anti-idiotypic antibodies: anti-IgE_(id) or anti-T_(HF) induces the formation in the animal of suppressor T-cells (i.e., T_(S) -cells). More specifically, the anti-idiotypic antibodies induce the formation of at least two sub-populations of suppressor T-cells, namely, T_(S).sbsb.1 and T_(S).sbsb.2 cells.

It is believed that the T_(S).sbsb.1 cells secrete a protein factor (T_(SF).sbsb.1) which binds the allergenic antigen itself by reaction with the allergenic determinant thereof.

Similarly, it is theorized that the T_(S).sbsb.2 cells secrete a protein factor T_(SF).sbsb.2 that binds with the idiotype receptors on the T_(H) (helper) cells and B-cells, thereby inhibiting the recognition by the T_(H) and B-cells of the antigen and, hence, also inhibiting the production thereby of allergenic antibody IgE.

Although any of the above-described anti-idiotypic antibodies or T-cell suppressor factors may be utilized to immunize a sensitive animal to allergic responses to Timothy grass antigen B or other grass antigens cross reactive therewith, it will be understood by those skilled in the art that the most advantageous immunization agents will comprise those derived from the animal(s) of the same species as that to be immunized. Since the immunization protocol usually comprises a series of immunizations over a long period of time, the more "foreign" the immunization protein, the more likely the prospect that the immune system of the animal will recognize the protein and produce antibodies directed against it. Accordingly, it is preferred to derive the anti-idiotypic antibody or suppression factor from animals or cells of animals of the same species as that to be immunized so as to produce non-immunogenic proteins. The whole suppressor cells (T_(S)) could not, of course, be administered to an animal of a species different from that from which the cells were derived since they would be rejected by the immunized animal.

It will also be understood by those skilled in the art that in the methods of production of anti-idiotypic antibodies, anti-IgE_(id) and anti-T_(HF), the second animal species immunized with IgE and T_(HF) to produce the antibodies should preferably be different from the first animal species in which the IgE and T_(HF) were produced in order to optimize production of the antibodies. In the case of producing anti-IgE_(id) it is also preferred to include the step of hyperimmunization when producing the IgE.

It will be further understood by those skilled in the art that although the examples set forth hereinbelow utilize affinity chromatography to achieve separation of the various protein fractions produced by the methods of the invention, any conventional method for separating proteins may be utilized.

For reasons not completely understood, animals immunized with photooxidized antigen B (Ox-AgB, prepared according to the methods described in U.S. Pat. No. 4,256,732, the disclosure of which is incorporated herein by reference) adsorbed in alum produce T_(H) cells from which T_(HF) can be derived. If, however, the Ox-AgB is incorporated in Freund's complete adjuvant (FCA) and administered to the animal, T_(S) cells are produced from which T_(SF) can be derived.

The steps of culturing the various proteins, antibodies or factors with cells may, of course, be conducted according to any standard culturing technique.

The references hereinbelow and in the claims to antigens D, D₁, D₂, D₃, etc. are to the antigen fragments which make up the mixture conventionally termed Timothy grass antigen B (see U.S. Pat. No. 4,256,732 and U.S. Pat. No. 4,140,679, the disclosures of which are also incorporated herein by reference).

In the above-described method for producing T_(S) cells by culturing the anti-idiotypic antibody with normal animal cells it is preferred to utilize normal spleen cells, etc. Where the T_(S) cells are to be employed for producing factors for use in humans it is preferred to utilize peripheral blood lymphocytes as the normal cells.

T-cells (T_(H) and T_(S)) may be derived from the spleens of immunized animals according to the method of Wysocki et al, Proc. Natl. Acad. Sci., Vol. 75, pg. 2844+ (1978), the disclosure of which is incorporated herein by reference.

The T_(SF) factor is preferably derived from the T_(S) cells by extraction. Preferably, a suspension of the T_(S) cells are repeatedly frozen and thawed to burst the cell walls and release the T_(SF) factor. The latter is preferably isolated from the mixture by affinity chromatography in an inert carrier substrate to which is bound antigen D₁.

Alternatively, the T_(SF) may be produced by culturing the T_(S) cells with either of the anti-idiotypic antibodies, anti-IgE_(id) or anti-T_(HF), or with antigen B at 37° C. for about 24 hours. The culture medium is preferably centrifuged and the cell-free supernatant subjected to affinity chromatography.

The T_(SF) fraction may be subjected to affinity chromatography to separate it into T_(SF).sbsb.1 and T_(SF).sbsb.2 fractions utilizing an inert carrier substrate to which is bound antigen D₁ (AgD₁) The adsorbed fraction is T_(SF).sbsb.1, which may be eluted with a variety of reagents (0.1 m molar antigen D, 3 molar KSCN in PBS buffer (pH 7.2), 0.1 molar glycine-HCl, pH3, etc.)

The non-adherent material contains T_(SF).sbsb.2 which may be isolated by affinity chromatography on an inert carrier substrate to which is bound T_(SF).sbsb.1. The adherent material is T_(SF).sbsb.2 which may be eluted with a variety of reagents (3 molar KSCN in PBS buffer (pH 7.2), 0.1 molar glycine-HCl, pH 3, etc.).

The T_(SF).sbsb.1 and T_(SF).sbsb.2 factors may be recovered from the eluants by dialysis against PBS buffer (pH 7.2) and concentrated by negative pressure to a convenient level.

The T_(SF).sbsb.1 and T_(SF).sbsb.2 fractions may be cultured with normal animal cells such as spleen cells, human peripheral blood lymphocytes, etc. at 37° C. for 4 days to produce T_(S).sbsb.1 and T_(S).sbsb.2 cells, respectively. The T_(S) cells may be recovered from the culture by affinity chromatography methods using antigen or T_(SF).sbsb.1 coated petri dishes or paper discs. The thus produced T_(S) cell population will provide 100% suppression of Timothy Ig response.

The pharmaceutical composition of the invention preferably contains from about 0.37 to 3.7 mg of either anti-IgE_(id), anti-T_(HF), T_(SF), T_(SF).sbsb.1 or T_(F).sbsb.2. The active ingredients may be compounded with any of the conventional adjuvants and carriers suitable for intravenous or subcutaneous administration of proteins.

To provide immunization against primary and secondary responses to Timothy grass antigen and cross reactive grass antigens, the composition should be administered every one to three months, preferably about once a month.

The invention is illustrated by the following non-limiting examples:

p EXAMPLE 1 Preparation of anti-IgE_(id) and anti-T_(HF)

The following abbreviations are employed herein: AgB, antigen B; AgD, antigen D; anti-E_(id), antiidiotypic antibody; anti-IgEm, rabbit anti-mouse timothy IgE, con a, concanavalin A; C.S. buffer, cacodylic-saline buffer; FCA, Freund's complete adjuvant; FCS, fetal calf serum; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; HF, helper factor; KLH, keyhold limpet hemocyanin; NRS, normal rabbit serum; OVA, ovalbumin; Ox-AgB, photooxidized antigen B; PBS, phosphate-buffered saline; PCA, passive cutaneous anaphylaxis; SF, suppressor factor; T_(H) cells, helper T cells; T_(HF), helper T cell factor; T_(S) cells, suppressor T cells; T_(SF), suppressor T cell factor; WST, extract of Timothy grass pollen.

Timothy grass pollen extract (WST), purified antigen D (AgD), AgB, and Ox-AgB were prepared as described in Malley, A., Baecher, L., Begley, D., and Forsham, A., Mol. Immunol. 16: 929 (1979); and Malley, A. and Harris, R. L., Jr., J. Immunol. 99: 825 (1967). (See also U.S. Pat. No. 4,256,732). Ovalbumin (OVA) was recrystallized three times. Antigen-B specific helper factor (T_(HF)) and suppressor factor (T_(SF)) were prepared according to the following methods.

Spleen cells from mice primed with Ox-AgB in alum were obtained 7 days after priming. A T cell enriched population was obtained by passage of these cells over nylon wool [Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3: 645 (1973)], and the non-adherent T cell population was cultured with an optimum concentration of Ox-AgB for 5 to 18 hours at 37° in 5% CO₂ in air. The cell-free supernatant (CFS) was removed by centrifugation, and AgB-specific T_(HF) was purified by affinity chromatography on Seph-AgD₁. The AgB-specific T_(HF) was eluted from the adsorbent with 3 M KSCN in PBS, pH 7.2. The recovered T_(HF) was applied to the Seph-AgD₁ adsorbent a second time, and greater than 95% of the protein bound was eluted with KSCN. As little as three micrograms of protein of the isolated AgB-specific T_(HF) injected intravenously with a limiting number (2.5×10⁶) of WST-primed B cells (anti-thy plus complement treated) induces a secondary IgE response with a titer of 3,000 in syngeneic x-irradiated (650 rads) recipients.

AgB-specific T_(SF) was obtained by sonication of spleen cells from mice primed with Ox-AgB in FCA. The CFS was collected by ultracentrifugation at 20,000 rpm for 20 min., and AgB-specific T_(SF) was isolated by passage twice over a Seph-AgD₁ affinity adsorbent (6). Ten micrograms (10 μg of protein) of AgB-specific T_(SF) completely suppresses a secondary anti-timothy IgE response.

LAF₁ mice, 8 to 12 weeks old, were immunized with WST as described in Fairchild, S., and Malley, A., J. Immunol. 115: 446 (1975); and Fairchild, S. and Malley, A., J. Immunol. 117: 2137 (1967). For the production of T_(H) cells, mice were immunized once intraperitoneally with 150 μg of Ox-AgB adsorbed on 1 mg of alum, and 7 days later single-cell preparations of their spleens were made as described in Fairchild et al, supra. Suppressor T cells were obtained from mice primed with 150 μg of Ox-AgB incorporated in FCA twice at 14-day intervals; the spleen cells were collected 7 days later.

Mice immunized with OVA received 10 μg injections of OVA protein adsorbed on 1 mg of alum, and the spleen cells were collected 7 to 10 days later.

Rabbit anti-helper factor was prepared by immunization of albino New Zealand rabbits with 150 μg of protein of affinity-purified AgB-specific T_(HF) in FCA distributed in 3 to 4 sites along the backs of the rabbits. Each rabbit received a booster injection of the same amount of T_(HF) in FCA at 12- to 18-day intervals over the next 50 days, and the animals were exsanguinated 7 days after the last injection of T_(HF).

Anti-IgE_(m) was prepared as described by Malley, A., Begley, D., and Forsham, A., Immunol. Commun. 6: 473 (1977), and the specific antibody (anti-E_(id)) was separated from anti-IgE_(Fc) (heavy-chain-specific) antibodies by affinity chromatography (described below).

Rabbit anti-T-helper factor and rabbit anti-E_(id) antisera were routinely passed over Sepharose rabbit anti-mouse Ig and Sepharose-fetal calf sera (Seph-FCS) adsorbents to remove any nonspecific antibodies present in these antisera.

Spleen cells or nylon wool enriched T cells from mice primed with Ox-AgB in alum or FCA, and OVA in alum, were cultured for 5 days at 37° C. in 5% CO₂ in air as described by Fairchild, supra, and Fairchild, S. and Malley, A., J. Immunol. 117: 2137 (1967). Cultures received pulses of ³ H-thymidine (1 μCi; 6.7 mmol/Ci, New England Nuclear, Gardena, Cal.) during the last 24 hours of culture. Upon termination of these cultures the cells were harvested by a minimultiple automatic sample harvester (Microbiological Associates, Los Angeles, Cal.), and harvested samples were counted on a Packard scintillation counter (Packard Instrument Company, Inc., Downers Grove, Ill.).

In experiments to block antigen-induced proliferation, whole spleen cells or nylon wool enriched T cells cultured with antigen (AgB or OVA) were preincubated for 90 min. at 37° C. with 750 μ1 of anti-T_(HF) serum, 150 μ1 of anti-Eid serum, or 750 μ1 of normal rabbit serum (NRS). The cells were washed 3 times with RPMI 1640 supplemented with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, 5% fetal calf serum (FSC) and penicillin-streptomycin (100 units/ml). These cells were diluted to 2 ml with the same media for a final dilution of 6×10⁶ cells/ml. One hundred microliters of these cell suspensions were added to each well of a Falcon micro-titer plate (Falcon Labware Division, Becton Dickinson, Oxnard, Cal.), and 5 μg of AgB (100 μ1), 0.5 μg of concanavalin A (con A) (100 μl), or 5 μg of OVA (100 μl were added and these mixtures were cultured for 5 days as described above.

The ability of anti-T_(HF) or anti-E_(id) to induce proliferation of T_(H) and T_(S) cells was determined by culturing nylon wool nonadherent T cells from mice primed with Ox-AgB in alum or cells from mice primed with Ox-AgB in FCA and enriched for T cells by the method of Wysocki et al, supra.

Spleen cells from mice immunized with WST were either passed over nylon wool to provide immune T cells or were treated with rabbit anti-thy 1.2 (1:40 final dilution at 4° C. for 1 hr) and guinea pig complement (1:10 final concentration) (Colorado Serum Company, Denver, Colo.) to provide immune B Cells.

Previous studies have demonstrated that intravenous injection of a limiting number of immune B cells (2.5×10⁶ cells) and 4×10⁶ immune T cells into X-irradiated (600 rads) syngeneic mice and subsequent challenge with 10 μg of WST in alum within 2 hours initiated a significant secondary IgE response. Serum IgE was measured by PCA in Sprague-Dawley rats according to the method of Fairchild, S. and Malley, A., J. Immunol. 115: 446 (1975), and IgG was measured by a modified radioimmunoassay as described by Malley, A, Begley, D. C., and Forsham, A., Int. Arch. Allergy Appl. Immunol., 62: 276 (1980).

The ability of anti-idiotypic antibodies to block or enhance T_(H) or B cell activity was determined through incubation of T_(H) or B cells (2×10⁷ cells) with 1 ml of anti-T_(HF) (diluted 1:2.5 in unsupplemented RPMI), 1 ml of anti-E_(id) (diluted 1:5 in unsupplemented RPMI) or 1 ml of NRS (diluted 1:2.5 in unsupplemented RPMI) for 90 min. at 37° C. The cell suspensions were washed 3 times in RPMI, mixed with the appropriate quantity of untreated immune T or B cells, and injected intravenously into X-irradiated (600 rads) syngeneic recipients.

The AgD fragment of timothy grass pollen was attached to Sepharose-AH-4B (Pharmacia Fine Chemicals, Piscataway, N. J.) as described by Malley, A., Baecher, L., and Begley, D., Immunochemistry 12: 551 (1975) and Malley, A., Baecher, L., and Begley, D., Dev. Biol. Stand. 29: 29 (1975). Antigen-B-specific T_(HF) and T_(SF) were partially purified by passage of the cell-free supernatant fluids containing either T_(HF) or T_(SF) over the Sepharose-AH-AgD (Sepharose-AgD) adsorbent. Nonadherent proteins were washed off with excess 0.01 M cacodylicsaline (C.S.) buffer, pH 7. Bound proteins were eluted with 3 M KSCN in C.S. buffer. Eluted proteins were dialyzed against phosphate-buffered saline (PBS) and concentrated by negative pressure.

Antigen B was attached to Sepharose 4B by the method of March et al, Ann. Biochem. 60: 149 (1978). The Sepharose-AgB adsorbent was treated with 3 ml of mouse anti-AgB IgE serum diluted to 10 ml with C.S. buffer. The IgE titer of this antiserum was 1:60,000 measured by PCA in Sprague-Dawley rats. When all of the diluted anti-AgB IgE serum was on the adsorbent, the flow was stopped and this suspension was incubated at room temperature for 2 hours. The adsorbent (10 ml of packed Sepharose) was washed with 500 ml of C.S. buffer to remove nonspecifi-cally bound protein. The first 100 ml of this wash was recovered and reconcentrated for recovery of unbound anti-AgB IgE antibodies. The Sepharose AgB-IgE complex was next treated with 0.02 M gluteraldehyde for 60 min. at room temperature to stabilize the bound IgE to the AgB. The Sepharose AgB-IgE adsorbent was washed with 250 ml of C.S. buffer, 50 ml of 3 M KSCN in C.S. buffer, 200 ml of distilled water, and 200 ml of each of the following reagents: 0.5 M Na₂ CO₃ -0.5 M NaCl, pH 10; 1 M NaAc-0.5 M NaCl, pH 4.0; 2 M urea-0.5 M NaCl; and C.S. buffer, pH 7.0.

The anti-IgE_(m) was separated into heavy-chain-specific (anti-IgE_(Fc)) and anti-Eid antibodies by passage over the Sepharose AgB-IgE adsorbent. The anti-IgE_(m) antibodies were precipitated with ammonium sulfate as described by Campbell et al, Methods in Immunology, W. A. Benjamin, Inc., New York, N.Y., p. 118 (1963), and the final precipitate was dialyzed extensively against C.S. buffer and concentrated by negative pressure. The protein concentration of the IgG preparation was determined spectrophometrically on the basis of the extinction coefficient of rabbit IgG, E₂₈₀ ^(1%) =14.5. Approximately 12 mg of protein (0.4 ml) of the IgG fraction of rabbit anti-IgE_(m) was applied to the Sepharose AgB-IgE fraction of rabbit anti-IgE_(m) was applied to the Sepharose AgB-IgE adsorbent in C.S. buffer. The nonadherent protein was eluted with C.S. buffer, and bound protein was eluted with 3 M KSCN in C.S. buffer. Upon dialysis and concentration, nonadherent and eluted fractions were assayed for their ability to neutralize AgB-specific IgE or OVA-specific IgE (a gift from Schering Company, West Germany). The fraction eluted from the Sepharose AgB-IgE adsorbent with KSCN was equally efficient in neutralizing AgB and OVA-specific IgE antibodies, and is therefore referred to as anti-IgE_(Fc). The fraction not adhering to the Sepharose AgB-IgE adsorbent was effective in neutralizing AgB-specific IgE but not OVA-specific IgE antibodies and is therefore referred to as anti-E_(id).

Attempts to conjugate affinity-purified AgB-specific T_(HF) or T_(SF) to Sepharose or affi-gel 10 (Bio-Rad Laboratories, Richmond, Cal.) and to have the conjugated factor retain its ability to react with antigen or anti-idiotypic antibodies were unsuccessful. However, pre-incubation of AgB-specific T_(SF) with soluble antigen for 1 to 2 hours at 37° C. prior to coupling with affi-gel 10 provided an insolubilized AgB-specific TSF adsorbent that could be used to purify anti-idiotypic antibody. Antigen-B-specific T_(SF) (500 μg of protein) was incubated with 25 mg of AgD at what was calculated to be about equal molar quantities. After incubation for 60 min. at 37° C. the solution was added to 12 g of affi-gel 10 equilibrated in 0.1 M NaHCO₃ (pH 8), and the suspension was stirred at room temperature for 6 hours and overnight at 4° C. The suspension was centrifuged at 2,500 rpm for 20 min. at 4° C. to remove the supernatant. The affi-gel matrix was washed in 0.1 M NaHCO₃ buffer (pH 8) and resuspended in 1 M ethanolamine (Mallinckrodt, Inc., St. Louis, Mo.) in 0.1 M NaHCO₃, ph 8. The suspension was stirred at room temperature for 1 hour and then washed 3 times in C.S. buffer. The washed adsorbent was treated with 3 M KSCN in C.S. buffer for 30 min. to remove AgD₁ bound to the T_(SF) and any free or weakly bound proteins. The excess KSCN was removed from the T_(SF) -affi-gel adsorbent by extensive washing in C.S. buffer and stored at 4° C. until ready for use.

Fetal calf serum (Grand Island Biological Company, Santa Clara, Cal.) was attached to Sepharose 4B by the method of March et al, supra. The Sepharose-FCS adsorbent was used to evaluate the specificity of anti-T_(HF) and anti-E_(id) binding to an AgB-specific T_(SF) affinity adsorbent.

The possibility that anti-T_(HF) serum contained anti-idiotypic antibody activity was examined with T cells from mice primed with Ox-AgB in alum preincubated with NRS, anti-T_(HF), or anti-E_(id) for 90 mi-. at room temperature. Preincubation of AgB-specific T_(H) cells with NRS did not reduce the ability of these cells to respond to either AgB or con A. Preincubation of these cells with either anti-T_(HF) or anti-E_(id) completely blocked their ability to respond with AgB, but did not reduce the level of ³ Hthymidine incorporation of these cells by con A. Specificity of the effect of anti-T_(HF) and anti-E_(id) was demonstrated by the failure of these antibodies to block either con A or OVA-induced proliferation responses with OVA/al-primed cells (Table 1). Spleen cells from nonimmunized mice pretreated with either anti-T_(HF) or anti-E_(id) did not respond when cultured with AgB or OVA, but gave normal Ievels of ³ H-thymidine incorporation with con A.

                                      TABLE 1                                      __________________________________________________________________________     Effect of pretreating ovalbumin- and photooxidized-                            antigen-B-primed cells with anti-antigen B idio-                               typic antisera upon the antigen-specific                                       lymphocyte transformation response.                                            Source of              .sup.3 H--thymidine incorporation.sup.b                 spleen cells                                                                            Treatment.sup.a                                                                      AgB     Con A   OVA                                             __________________________________________________________________________     Ox--AgB-primed.sup.c                                                                    NRS   47,521 ± 1,125                                                                      39,758 ± 1,102                                                                      1,075 ± 500                                           anti-T.sub.HF                                                                         910 ± 310                                                                          40,150 ± 1,275                                                                      1,210 ± 250                                           anti-E.sub.id                                                                        1,170 ± 450                                                                         39,927 ± 1,151                                                                       975 ± 390                                   Ova-primed.sup.d                                                                        NRS    975 ± 250                                                                          37,521 ± 1,395                                                                      32,175 ± 1,161                                        anti-T.sub.HF                                                                        1,210 ± 300                                                                         39,357 ± 1,510                                                                      35,193 ± 1,710                                        anti-E.sub.id                                                                        1,157 ± 291                                                                         39,771 ± 2,100                                                                      34,710 ± 1,423                               __________________________________________________________________________      .sup.a Nylon wool enriched T cells from mice primed with either OVA or         Ox--AgB were preincubated for 90 min. at 37° C. with 750 μl of       heatinactivated antiT.sub.HF, NRS, or 150 μl of antiE.sub.id. The cell      were washed 3 times wit h 5%FCS-supplemented RPMI 1640 media, and              suspended to a final volume of 2 ml.                                           .sup.b Cell suspensions, at a final concentration of 6 × 10.sup.6        cells/ml, were cultured with 100 μl of each cell preparation and the        optimal concentrations of AgB (10 μg of protein), con A (0.5 μg of       protein). The values reported a re the means of triplicate cultures ±       SD and are corrected for background.                                           .sup.c Animals were immunized with 150 μg of Ox--AgB adsorbed on 1 mg       of alum. Spleens were collected 7 days later, and 2 × 10.sup.8 cell      were passed over nylon wool columns.                                           .sup.d Animals were immunized with 10 μg of OVA adsorbed on 1 mg of         alum. Spleens were collected 7 days later, and 2 × 10.sup.8 cells        were passed over nylon wool columns.                                     

The direct proliferative effect of anti-T_(HF) and anti-E_(id) antisera upon AgB-specific T_(H) and T_(S) cells is shown in FIG. 1. Nylon wool nonadherent T cells from mice primed with Ox-AgB in alum provided a source of T_(H) cells. T cells enriched by depleting B cells on petri dishes coated with goat anti-mouse Ig (provided by National Institute for Medical Research, Mill Hill, England) from mice primed with Ox-AgB in FCA provided a source of T_(S) cells. Both T_(H) H and T_(S) S populations contained less than 5% cells that stained with fluorescein-anti-mouse Ig (Micro-biological Associates, Los Angeles, Cal.). Both T_(H) and T_(S) cells show significant levels of ³ H-thymidine incorporation at concentrations of anti-T_(HF) and anti-E_(id) that are 20 to 50 times lower than that used to block proliferation of T_(H) cells (Table 1).

Since anti-T_(HF) and anti-E_(id) initiated significant levels of ³ H-thymidine incorporation with AgB-specific T_(H) cells, it was determined if the action of anti-T_(HF) and anti-E_(id) antisera upon either T_(H) cells or immune B cells (primed with WST and treated with anti-thy 1.2 and complement) would enhance the secondary AgB-specific IgE response in X-irradiated recipients (Table 2). Immune T cells (nonadherent to nylon wool) from WST-primed mice mixed with immune B cells at a limiting ratio gave a good secondary IgE response. Preincubation of either T_(H) or B cells with NRS did not alter the secondary IgE response in the irradiated recipients. On the other hand, preincubation of either T_(H) or B cells with anti-T_(HF) or anti-E_(id) antisera significantly enhanced the secondary IgE response in the irradiated recipients. The level of enhanced antibody formation obtained by pretreatment with anti-E_(id) was slightly less than that achieved with OVA-primed T cells (nylon wool nonadherent) or B cells (anti-thy and complement) did not alter their immune response in X-irradiated syngeneic recipients. The AgB-specific IgG antibody response in all of the groups tested was less than the sensitivity of the radioimmunoassay (100 ng/ml).

                  TABLE 2                                                          ______________________________________                                         Enhancement of the antigen-B-specific IgE response                             by pretreatment of immune cell populations                                     with anti-idiotype.                                                                    Cell population.sup.b                                                                       Ratio of cells                                            Treatment.sup.a                                                                        treated      given recipients.sup.c                                                                      IgE response.sup.d                           ______________________________________                                         None    --           T/B            600                                                             4 × 10.sup.6 /2.5 × 10.sup.6                  NRS     T                           600                                        anti-T.sub.HF                                                                          T            4 × 10.sup.6 /2.5 × 10.sup.6                                                    2,800                                        anti-E.sub.id                                                                          T                         1,600                                        NRS     B                           600                                        anti-T.sub.HF                                                                          B            4 × 10.sup.6 /2.5 × 10.sup.6                                                    3,200                                        anti-E.sub.id                                                                          B                         1,600                                        ______________________________________                                          .sup.a Cell populations (T or B cells) were treated with 1.5 ml of             heatactivated NRS or antiT.sub.HF and 1.0 ml of antiE.sub.id per 2 .times      10.sup.7 cells. These mixtures were rocked at 37° C. for 90 min.        and centrifuged at 1,000 rpm for 10 min.; the supernatant was discarded.       The cell pellets were resuspended in PBS and washed 2 more times with PBS      before being resuspended to a final volume of 2 × 10.sup. 7 per ml.      .sup.b Spleen cells from mice immunized with 10 μg of protein of WST        adsorbed on 1 mg of alum at 21day intervals were collected on day 28.          Singlecell suspensions were prepared; portions of these cells (2               ×10.sup.8 cells) were passed over n ylon wool columns and represent      immune T cells (not adherent to nylon wool). The remaining cells, treated      with antithy 1.2 (L:10) and complement (1:10), represent immune B cells.       .sup.c Syngeneic recipients were Xirradiated (600 rads) and received           intravenous injections of 4 × 10.sup.6 T cells and 2.5 ×           10.sup.6 B cells. All recipients were challenged within 2 hrs. of this         injection with 10 μg of WST.                                                .sup.d Passive cutaneous anaphylaxis responses were determined in              duplicate in SpragueDawley rats. The values represent the mean reactions       observed in at least 2 rats. At least 3 animals per group made up the          serum pool tested, and blood was colle cted 7, 10 and 14 days after cell       transfers. The titer reported represents the mean titer of the maximum         response (day 7) observed.                                               

Purification of anti-idiotypic antibodies by affinity chromatography was achieved with an AgB-specific T_(SF) -affi-gel adsorbent (affi-gel 10_(SF)). Fractions of anti-T_(HF) or anti-E_(id) eluted from the affi-gel-T_(SF) with KSCN in PBS (pH 7.2) retained their ability to initiate ³ H-thymidine incorporation when cultured with spleen cells from mice primed with either WST or Ox-AgB in alum (Table 3). These same fractions were inactive (background levels of ³ H-thymidine incorporation) in cultures with normal spleen cells (data not shown). The elute fraction of NRS (1:5) applied to affi-gel-T_(SF) adsorbent was also inactive. The anti-idiotypic antibodies present in anti-T_(HF) antiserum did not bind nonspecifically to either a Sepharose-FCS adsorbent (Table 3) or an affi-gel adsorbent saturated with 1 M ethanolamine.

                  TABLE 3                                                          ______________________________________                                         Purification of anti-idiotypic antibodies on an                                antigen-B-specific T suppressor factor affi-gel                                affinity adsorbent.                                                                                         Volume .sup.3 H--thymidine                                                     tested incorporation.sup.b                        Sample   Adsorbent Fraction.sup.a                                                                           (μl)                                                                               (CPM ± SD)                              ______________________________________                                         anti-T.sub.HF (1:5)                                                                     Seph-FCS  column    10     13,160 ±                                                                    875                                                           pass      50     28,190 ±                                                                    2,045                                                                   100    21,200 ±                                                                    2,127                                      anti-T.sub.HF (1:5)                                                                     Seph-FCS  eluate    10     322 ± 362                                                            50     590 ± 319                                                            100    965 ± 475                               normal rabbit                                                                           affi-gel  eluate    10     375 ± 290                               serum (1:5)                  50     497 ± 418                                                            100    921 ± 510                               anti-T.sub.HF (1:5)                                                                     affi-gel T.sub.SF                                                                        eluate    10     26,009 ±                                                                    2,153                                                                   50     36,240 ±                                                                    1,741                                                                   100    29,959 ±                                                                    1,915                                      anti-T.sub.HF (1:5)                                                                     affi-gel T.sub.SF                                                                        column    10     435 ± 275                                                  pass      50     543 ± 295                                                            100    595 ± 280                               anti-E.sub.id (1:5)                                                                     affi-gel T.sub.SF                                                                        eluate    10     8,975 ± 1,201                                                        50     15,375 ±                                                                    975                                                                     100    11,811 ±                                                                    917                                        anti-E.sub.id (1:5)                                                                     affi-gel T.sub.SF                                                                        column    10     515 ± 195                                                  pass      50     621 ± 250                                                            100    710 ± 235                               ______________________________________                                          .sup.a Oneto fivemilliliter portions of antiT.sub.HF, antiE.sub.id, or         normal rabbit antiserum were applied to 10to 15ml packed volumes of the        indicated affinity adsorbents. The column pass (nonadherent) and eluate        (adherent) fractions wer e dialyzed against PBS, pH 7.2, and concentrated      by negative pressure back to their original volume.                            .sup.b Cell suspensions, at a final concentration of 6 × 10.sup.6        /ml, were cultured with 100 μl and the indicated volumes of antisera.       RPMI 1640 medium supplemented with 5% fetal calf serum was added to each       well for a final volume of 200 μ l/well. The values reported represent      the means of triplicate cultures ± SD and are corrected for background                                                                               

EXAMPLE 2 Regulation of primary and secondary anti-AgB-specific IgE responses with anti-idiotypic antibodies.

The data hereinbelow indicates that both anti-idiotypic antibodies suppress AgB-specific IgE responses, the suppression lasting for at least 35 days, and being mediated by a thy 1⁺ Lyt 1⁻ 23⁺ T cell.

Timothy pollen extract (WST), purified antigen B (AgB), phooxidized antigen B (Ox-AgB), and AgB-specific T_(SF) were prepared as described above.

CBA/Ca by C57BL6 F₁ mice and Sprague-Dawley rats were bred under specific pathogen-free conditions at the National Institute for Medical Research, Mill Hill, London. In most experiments male mice 9 to 12 weeks old and rats weighing 200 to 250 g were used.

Animals treated with WST were injected intraperitoneally (i.p.) with 10 μg of protein of WST adsorbed on 1 mg of alum on day 0. Animals given a secondary antigen challenge were treated with the same dose of WST in alum on day 21.

Normal mice were injected with affinity-purified normal rabbit IgG (nRGG), anti-T_(HF), or anti-IgE_(id) from 0.1 to 10 μg of protein per day for 3 successive days. Twenty-four hours later immunized animals were primed with 10 μg of protein of WST adsorbed on 1 mg of alum and 21 days later these animals were given a secondary boost with the same dose of WST.

The indiction of anti-ovalbumin IgE responses was achieved by injecting animals with 10 μg of protein ovalbumin (OVA) absorbed on 1 mg of alum.

The Fab₂ fragments of anti-T_(HF) were prepared by pepsin digestion of the intact anti-T_(HF) antibody at pH 4.2 [Williams, C. A., and Chase, M. W., Academic Press, 1: 422 (1967]. The digest was then passed over a protein-A-Sepharose adsorbent to remove intact undigested antibodies and Fc fragments present in the digest. The effluent not adherent to the protein-A-Sepharose adsorbent was concentrated by negative pressure against PBS buffer to a concentrate of 0.84 mg of protein/ml. Normal rabbit IgG was purified from normal rabbit serum by passage of rabbit serum over a protein-A-Sepharose adsorbent. The sample bound to the adsorbent was eluted with 3 M KSCN in PBS buffer, pH 7.2. The eluted fraction was concentrated by negative pressure against PBS buffer, pH 7.2, to a final concentration of 2.5 mg of protein/ml, and gave a single precipitin band against anti-normal rabbit sera and anti-rabbit IgG.

The anti-Thy 1.2 antiserum was monoclonal antibody prepared at the National Institute for Medical Research (NIMR), Mill Hill, London, England (designated NIMR-1), and goat anti-mouse Fab antisera was obtained from NIMR, Mill Hill, London.

Spleen cells from animals treated with either normal rabbit IgG or anti-T_(HF) were removed under aseptic conditions, washed in PBS, and varying amounts of these cells were injected intravenously into recipient animals. All recipient animals had been primed 20 days earlier with 10 μg of protein of WST adsorbed on 1 mg of alum, and received a secondary boost of the same dose of antigen within 24 hours after cell transfer. In some experiments donor cells were treated with either normal rabbit serum or anti-Thy 1.2 antiserum and complement before the washed cells were injected into recipient animals.

The method of Wysocki et al, supra, was used to obtain a population of enriched T cells from spleen cell populations. Briefly, plastic-coated petri dishes were coated with anti-mouse Fab antibody and 3×10⁷ spleen cells (in 3 ml) were incubated per dish at 4° C. for 60 min. Nonadherent cells were removed by Pasteur pipette and the coated dishes washed with 7 ml of cold buffer. The combined wash and initial aliquot of nonadherent cells were washed several times in cold buffer and represent a T-enriched cell population. Analysis of the nonadherent cell population indicated that this cell population was between 2 and 5% Ig⁺ when examined by fluorescein-conjugated anti-mouse Ig sera, and at least 95% Thy-1+ when stained with fluorescein-conjugated anti-Thy-1 antisera.

The serum IgE titers were measured by passive cutaneous anaphylaxis (PCA) [Moto et al, Life Science, 8: 813 (1969) and Fairchild et al, J. Immunol. 115: 446 (1975)] with 500 μg of WST in phosphate-buffered saline (PBS) as the antigen challenge. The PCA titer was measured in duplicate in Sprague-Dawley rats and is expressed as the reciprocal of the highest dilution of serum yielding a 5-mm (diameter) bluing reaction.

Sepharose adsorbents coupled with normal mouse serum (NMS), the F(ab)₂ fragments of mouse IgG, and AgB-specific T_(HF) were described above in Malley et al, Immunol. Commun. 6: 473 (1977) Protein-A-Sepharose adsorbent was purchased from Pharmacia (Piscataway, N.J.).

Pretreatment of normal mice with nRGG for 3 successive days prior to immunization with 10 μg of protein of WST absorbed on 1 mg of alum did not alter the normal primary or secondary response to WST (Table 4). On the other hand, pretreatment with either anti-T_(HF) or anti-IgE_(id) completely suppressed a primary WST response, and yielded a dose-related suppression of the secondary WST response (Table 5). The suppression induced by anti-T_(HF) or anti-IgE_(id) pretreatment persists at least 35 days. The observed suppression could be due either to the induction of a population of suppressor T cells or by antibody feedback suppression. In experiments to distinguish between these two possibilities, we found that cells from mice injected with nRGG and treated with either NRS or anti-thy 1.2 and C' did not alter the secondary anti-AgB IgE response. However, cells obtained from mice injected with anti-T_(HF) and treated with NRS and C' suppressed the secondary anti-AgB IgE response, but treatment of these cells with anti-thy 1.2 and C' completely abrogated the observed suppression. This suggests that the reduction in anti-AgB IgE antibody by anti-T_(HF) treatment was mediated by a population of suppressor T (T_(S)) cells.

                  TABLE 4                                                          ______________________________________                                         The effect of pretreatment of normal mice -with anti-idiotypic antibody        upon the                                                                       primary and secondary anti-timothy IgE responses.                                           Primary       Secondary                                                  Dose  IgE response.sup.b                                                                           IgE response.sup.b                                  Treatment.sup.a                                                                         g/day   day 7     day 19                                                                               day 7   day 14                                ______________________________________                                         normal   10.0    50        100   800     600                                   rabbit IgG                                                                     anti-T.sub.HF                                                                           0.1     --        --    400     200                                   anti-T.sub.HF                                                                           1.0     --        --    200     100                                   anti-T.sub.HF                                                                           10.0    --        --    200     100                                   anti-IgE.sub.id                                                                         0.1     --        --    400     200                                   anti-IgE.sub.id                                                                         1.0     --        --    400     100                                   anti-IgE.sub.id                                                                         10.0    --        --    200     100                                   ______________________________________                                          .sup.a Affinity purified normal rabbit IgG (nRGG), antiT.sub.HF, or            antiIgE.sub.id was injected intravenously at the indicated doses for thre      successive days (-3 to -1) into six normal mice per group. All animals         were challenged with 10 μ g of protein of WST adsorbed on 1 mg of alum      on day 0, and given a secondary boost with the same dose of WST on day 21      .sup.b IgE responses were measured by passive cutaneous anaplylaxis (PCA)      in SpragueDawley rats. All titrations were done in triplicate and the          values represent the reciprocal of the mean of the lowest dilution giving      a 5 × 5 positive reaction .                                        

                  TABLE 5                                                          ______________________________________                                         Evidence of anti-idiotypic antibody induction                                  of T cells that suppress a secondary anti-timothy IgE Response                          Donor cell                                                                               IgE response.sup.c                                          Immunization.sup.a                                                                        treatment.sup.b                                                                            Day 7     11   14                                       ______________________________________                                         normal     NRS + C'    1600      1200 800                                      rabbit IgG anti-Thy + C'                                                                              1600      1200 800                                      anti-T.sub.HF                                                                             NRS + C'     200       100 100                                                 anti-Thy + C'                                                                              1600      1200 800                                      ______________________________________                                          .sup.a Normal mice were injected with 10 μg of protein of either norma      rabbit IgG or affinity purified antiT.sub.HF daily for 3 days. On day 4        the spleens were removed and singlecell suspensions made.                      .sup.b Cell suspensions were treated with equal volumes of either normal       rabbit sera (NRS) or antiThy 1.2 sera and guinea pig complement (C') at a      1:3 dilution for 1 hour at 37° C. Donor cells, after treatment wit      NRS or antiThy 1.2 and C ', were washed twice with PBS and 2.5 ×         10.sup.7 cells were injected intravenously into six recipient mice per         group. Recipients had been primed 20 days earlier with 10 μg of protei      of WST on 1 mg of alum. Twentyfour hours after cell tra nsfer, all mice        received a secondary WST challenge (10 μg of protein in alum).              .sup.c IgE responses were measured by PCA in SpragueDawley rats. All           titrations were done in duplicate and the values represent the reciprocal      of the mean of the lowest dilution giving a 5 × 5 mm positive            reaction.                                                                

Further evidence that the observed suppression is mediated by a T_(S) cell population is shown by Table 6. Animal given cells from mice treated with nRGG and nonadherent to goat anti-mouse-Ig-coated petri dishes did not alter a secondary anti-AgB response. Animals given cells from mice treated with anti-T_(HF) and nonadherent to goat anti-mouse-Ig-coated petri dishes significantly suppressed (>90%) a secondary anti-AgB IgE response, and as little as 10⁶ of the enriched T cells transferred into primed recipients resulted in a 75% suppression of the secondary response.

                  TABLE 6                                                          ______________________________________                                         Titration of cells non-adherent on goat                                        anti-mouse Ig--(Fab)-coated petri dishes.                                                                   IgE response.sup.c                                        Number of  Number of Day                                               Immunization.sup.a                                                                       cells transferred                                                                           recipients.sup.b                                                                         7     14                                      ______________________________________                                         anti-T.sub.HF                                                                            4 × 10.sup.7                                                                          6         200   0                                                 10.sup.7     6         300   0                                                 5 × 10.sup.6                                                                          6         400   0                                                 10.sup.6     6         400   0                                                 5 × 10.sup.5                                                                          4         800   200                                               10.sup.5     4         1,600 400                                     nRGG.sup.d                                                                               3 × 10.sup.7                                                                          6         1,600 400                                     ______________________________________                                          .sup.a Animals were injected intravenously with 10 μg of protein of         either antiT.sub.HF or nRGG daily for three days. Twentyfour hours later       spleens were removed and applied to goatanti-mouse Ig--(Fab)coated plates      at 4° C. Nonadherent cells were recovered, washed twice with PBS,       and resuspended in PBS.                                                        .sup.b All recipients were primed twenty days earlier with 10 μg of         protein of WST adsorbed on 1 mg of alum. The indicated number of cells         nonadherent to the goatanti-mouse Ig plates were injected intravenously        and within 24 hours all recipients were given a secondary WST challenge.       .sup.c IgE response was measured in duplicate by PCA in SpragueDawley          rats. The mean titer is reported.                                              .sup.d nRGG  normal rabbit gamma globulin.                               

Specificity of the suppression induced by anti-T_(HF) treatment is shown by Table 7. Animals treated with anti-T_(HF) and immunized with 10 μg of ovalbumin adsorbed on 1 mg of alum have the identical primary IgE response as control animals treated intravenously with saline and then immunized with ovalbumin.

                  TABLE 7                                                          ______________________________________                                         Effect of anti-T.sub.TF antibody treatment upon                                the induction of anti-ovalbumin IgE formation.                                                  Num-    IgE Response.sup.c                                    Immuni-          ber of  Day                                                   zation.sup.a                                                                          Treatment.sup.b                                                                          animals 7        10  14                                       ______________________________________                                         OVA    none      4       800(600-1200)                                                                           --  400(100-600)                             OVA    anti-T.sub.HF                                                                            4       800(600-1200)                                                                           --  400(100-600)                             ______________________________________                                          .sup.a All animals were immunized intraperitoneally with 10 μg of           ovalbumin adsorbed on 1 mg of alum.                                            .sup.b Animals were injected intravenously with either saline or 10 μg      of protein of antiT.sub.HF daily for three days. On the next day all           animals were immunized with ovalbumin.                                         .sup.c IgE responses were measured in duplicate by PCA in SpragueDawley        rats. The mean titers and the range (in parenthesis) of responses are          indicated.                                                               

Table 8 compares the ability of anti-T_(HF), anti-IgE_(id), and the F(ab)₂ fragment of anti-T_(HF) to induce T_(S) cells. Although anti-IgE_(id) is as effective as anti-T_(HF) in inducing T_(S) cells, the F(ab)₂ fragment of anti-T_(HF) does not significantly induce T_(S) cells.

                  TABLE 8                                                          ______________________________________                                         In vivo induction of supressor T cells                                         by anti-idiotypic antibody.                                                            Number    Number                                                       Immuni- of cells  of re-   IgE response.sup.c                                  zation.sup.a                                                                            transferred                                                                             cipients.sup.b                                                                          0  100  200  400  600  800  1200                    ______________________________________                                                                    1600                                                anti-T.sub.HF                                                                          3 × 10.sup.7                                                                       15                                                           anti-IgE.sub.id                                                                        5 × 10.sup.7                                                                       6                                                            anti-   5 × 10.sup.7                                                                       8                                                            T.sub.HF (Fab).sub.2.sup.d                                                     anti-T.sub.HF                                                                          .sup. 3 × 10.sup.7.spsp.e                                                          6                                                            nRGG.sup.f                                                                             2.5 × 10.sup.7                                                                     12                                                           ______________________________________                                          .sup.a Animals injected intravenously daily for 3 days with 10 μg           anitT.sub.HF, 50 μg antiE.sub.id, or 80 μg of antiT.sub.HF               (Fab).sub.2. On day 4 spleens were removed, single cell suspension             prepared, and the indicated number of viable cells injected into               recipients.                                                                    .sup.b Recipients were primed with 10 μg of WST in alum 20 days             earlier, and all animals were given a secondary challenge within 24 hours      after receiving cell transfers.                                                .sup.c IgE response measured in duplicate in SpragueDawley rats. The mean      of the maximum responses (day 7) is reported, and the range of responses       is indicated by the bars.                                                      .sup.d Pepsin digest of antiT.sub.HF and passed over a SepharoseProtein A      adsorbent.                                                                     .sup.e AntiThy 1.2 and C' treated.                                             .sup.f Normal rabbit IgG eluted from Sepharose Protein A adsorbent and         injected into donor mice at 10 μg of protein per day for 3 days.      

At the concentrations used, both anti-idiotypic antibodies (anti-T_(HF) and anti-IgE_(id)) completely suppressed primary anti-AgB IgE responses, and up to 75% of the secondary response in recipients immunized with WST adsorbed on alum (Table 4). The duration of the anti-idiotypic induced suppression was fairly long lived, lasting more than 35 days. The observed suppression is mediated by a T cell population as indicated by abrogation of suppression by treating cells from anti-idiotypic antibody treated animals with anti-thy 1.2 antisera and complement, and the enrichment of the suppressor cell populations in the fraction nonadherent to anti-mouse-Ig-coated petri dishes (Tables 5 and 6). The failure of mice treated with anti-idiotypic antibody prior to immunization with ovalbumin to yield antibody titers different from control mice (treated with saline or nRGG) not only indicates the specificity of anti-idiotype-induced suppressor T cells, but also argues against the suppression being an anti-immunoglobulin-induced feedback mechanism (Table 7).

The failure of the F(ab)₂ fragment of anti-T_(HF) antibody to induce a suppressor cell population could reflect a difference in half-life of the intact antibody in the circulation compared to the fragment. It was attempted to overcome this potential problem by using up to eight times (80 μg of protein) the amount of the F(ab)₂ fragment compared to the intact (10 μg of protein) anti-idiotypic antibody without success. The F(ab)₂ fragment of anti-T_(HF) antibody retained its full combining site activity as demonstrated by its ability to completely block the antigen-induced PCA activity of sera from mice having an AgB-specific IgE titer of 30,000. Others have similarly reported that F(ab)₂ fragments of anti-idiotypic antibodies are ineffective immunosuppressants [Rowley, D. A., Fitch, F. W., Stuart, F. P., Kohler, H., and Cosenza, H., Science 181: 1133 (1973); Fitch, F. W. Prog. Allergy 19: 195 (1975); Forni, L., and Pernis, B., Membrane Receptors of Lymphocytes, American Elsevier, N.Y., (1975); Sidman, C. L., and Unanue, E. R., J. Exp. Med. 144: 882 (1976); Pierce, S. K., and Klinman, N. R., J. Exp. Med. 146: 509 (1977); and Moretta, L., Mingari, M. C., and Romanzi, C. A., Nature 272: 618 (1978)]. Recently, several investigators have suggested that B cells may play an important role in the activation of T cell function [Zubler, R. H., Benacerraf, B., and Germain, R. N., J. Exp. Med. 151: 677 (1980); Zubler, R. H., Benacerraf, B., and Germain, R. N., J. Exp. Med. 151: 681 (1980); L'age-Stehr, J., Teichmann, H., Gershon, R. K. and Cantor, H., Eur. J. Immunol. 10: 21 (1980); and Black, S. J. and Herzenberg, L. A., J. Exp. Med., 150: 174 (1979)]. The failure of the F(ab)₂ fragment of anti-T_(HF) antibody to induce a suppressor cell population may reflect that either macrophages or B cell processing via their F_(c) receptors is needed. Alternatively, T cells with F_(c) receptors [Setcavage, T. M. and Kim, Y. B., J. Immunol. 124: 553 (1980)] may play an important role in the induction of suppressor T cells.

Characterization of the suppressor cell induced by anti-idiotypic antibody indicates that it is a Thy 1+ Lyt 1⁻ 23⁺ T cell. Preliminary studies demonstrated that the factor extracted from spleen cells treated with anti-T_(HF) antibody and nonadherent to anti-mouse-Ig-coated petri dishes binds to idiotype-positive affinity adsorbents (Sepharose-antigen-B-specific suppressor factor) suggesting that the suppressor T cell induced is a T_(S).sbsb.2 suppressor cell population.

EXAMPLE 3 Induction of suppressor T-cells (T_(S))

CBA/CaxC57BL6 F₁ mice and Sprague-Dawley rats were bred under specific pathogen-free conditions at the National Institute for Medical Research (NIMR), Mill Hill, London. In most experiments male mice 9 to 12 weeks old and rats weighing 200 to 250 g were used.

Animals treated with WST were injected intraperitoneally (i.p.) with 10 μg of protein of WST adsorbed on 1 mg of alum on day 0. Animals were given a secondary antigen challenge with the same dose of WST in alum on day 21.

Animals treated with ovalbumin (OVA) were injected i.p. into 10 μg of protein of OVA adsorbed on 1 mg of alum on day 0, and were given a secondary antigen challenge with the same dose of OVA in alum on day 21.

Normal rabbit IgG (nRGG) was prepared by passing normal rabbit serum over a Sepharose-protein A adsorbent, and after washing the adsorbent with seven columns (about 100 ml) of buffer the bound rabbit IgG was eluted with 3 M KSCN in phosphate-buffered saline (PBS), pH 7.2. Gel-diffusion analysis of the isolated fraction gave a single precipitin band against anti-rabbit IgG and anti-whole rabbit sera.

Anti-Lyt 1 and anti-Lyt 2 antisera were obtained from NIMR, Mill Hill, London, and were derived from monoclonal cell lines obtained from Stanford University, Stanford, Calif. Anti-thy 1.2 antisera was obtained from NIMR, Mill Hill, London, and was derived from a monoclonal cell line designated NIMR-1.

Normal spleen cells were removed by asceptic methods, washed three times with sterile RPMI-1640 media, and diluted in complete media to 10⁷ viable cells/ml. Complete media consisted of RPMI-1640 medium supplemented with 25 mM HEPES, 12 mM NaHCO₃, sodium pyruvate (1 mM), 2-mercaptoethanol (2×10⁻⁵ M), 10% fetal calf serum (FCS), L-glutamine (2 mM), nonessential amino acids, and penicillin/streptomycin (100 U and 10 μg/ml). Cultures consisted of 107 normal spleen cells (1 ml of cell suspension) in each mini-Marbrook chamber (Hendley Engineering, Loughton, England) placed in 100×15 mm sterile Falcon tissue culture dishes containing 10 ml of complete media. The cell suspensions were maintained in the mini-Marbrook chambers by washed and boiled dialysis tubing cut to the appropriate size. All cultures were maintained for 4 days in a moist atmosphere containing 10% CO₂, 7% O₂, and 83% N₂ at 37° C. Upon termination of the cultures, the cells are washed three times, counted, and various numbers of viable cells were injected intravenously into mice primed 20 days earlier with 10 μg of protein of WST adsorbed on 1 mg of alum. All recipients were given a secondary antigen challenge within 24 hours with the same dose of WST in alum.

Serum IgE titers were determined by passive cutaneous anaphylaxis (PCA) in Sprague-Dawley rats. All titrations were done in duplicate, and the mean titers and range of response (in parenthesis) are reported.

Serum IgG titers were analyzed by a radioimmunoassay using WST-conjugated paper disks and the level of timothy-specific IgG antibodies was less than 100 ng of IgG.

An indirect microtiter plate ELISA to measure AgB-specific T_(SF).sbsb.1 and T_(SF).sbsb.2 was developed according to the method of Voller et al [Manual of Clin. Imm. Ed. by N. Rose et al, Pg. 506 (1976) Amer. Soc. Microbiol.] with some modification. Rabbit anti-T_(HF) and sheep anti-rabbit IgG antibodies were prepared as above. Sheep anti-rabbit IgG (0.8 mg) in 0.5 ml was dialyzed extensively with PBS, pH 7.2, at 4° C. with alkaline phosphatase (Miles Laboratories, Inc., Elkhart, Ind.) and conjugated with 0.2% gluteraldehyde. Excess gluteraldehyde was removed by additional dialysis against PBS, pH 7.2, at 4° C., and the final conjugate was mixed human serum albumin (3% final concentration), and sodium azide (0.02% final concentration). Flat-bottomed micro ELISA polystyrene plates (Dynatech Laboratories, Inc., Alexandria, Va.) were coated with 200 μl of affinity purified AgB-specific T helper factor (T_(HF)) containing 475 μg of protein/ml by incubation at 37° C. for 3 hours. The plates were washed with a PBS-Tween-20 buffer and incubated overnight at 4° C. with 50 μg (150 μl) of affinity purified anti-T_(HF). The plates were washed with PBS-Tween-20, and incubated for 3 hours at 37° C. with sheep anti-rabbit IgG enzyme conjugate (1/25 dilution). The plates were washed with PBS-Tween-20, then substrate buffer, and finally incubated at 37° C. for 90 min. with p-nitrophenyl phosphate (Sigma 104, St. Louis, Mo.) (1 mg/ml). The reaction was stopped by the addition of 50 μl of 3 M NaOH and the developed color was measured spectrophotometrically at 405 nm.

Antigen B-specific T_(SF) was quantitated by inhibition of anti-T_(HF) binding to microtiter plate bound T_(HF). Anti-T_(HF) (50 μg of protein) is incubated with varying concentrations of T_(SF) for 2 hours at room temperature before adding this mixture to the T_(HF) -coated microtiter plates and incubated overnight at 4° C. The soluble factor extracted from T_(S).sbsb.2 cells, anti-idiotype factor (T_(SF).sbsb.2) is determined by addition of affinity purified material (Sepharose-T_(SF)) to T_(HF) bound microtiter plates for 2 hours at room temperature prior to the addition of anti-T_(HF) and incubation overnight at 4° C.

Cultures of normal spleen cells with anti-idiotypic antibody (anti-T_(HF) and anti-IgE_(id)) yield a population of T_(S) cells that significantly suppress a secondary IgE response in recipients (Table 9). Animals similarly treated with cells cultured with nRGG have the same secondary IgE response as animals given only a primary and secondary antigen challenge. The suppression induced by spleen cells cultured with anti-idiotypic antibody (anti-Id) is lost upon treatment of these cells with anti-thy 1 antisera and guinea pig complement (C) suggesting that the observed suppression is due to suppressor T cells. Anti-Id between the concentrations of 10 to 100 μg appears to be equally effective in inducing a population of suppressor T (T_(S)) cells (Table 9).

                  TABLE 9                                                          ______________________________________                                         Anti-idiotypic antibody induction of                                           suppressor T cells in vitro.                                                                        No. of  IgE response.sup.c                                Dose       No. of cells                                                                             recipi- Day                                               Treatment.sup.a                                                                        (μg)                                                                               transferred.sup.b                                                                        ents    7     10   14                                 ______________________________________                                         anti-T.sub.HF                                                                          10     4 × 10.sup.6                                                                        6    50(0-100)                                                                              0    0                                          25     6 × 10.sup.6                                                                        9    50(0-100)                                                                              0    0                                          50     8 × 10.sup.6                                                                       15    200(50-400)                                                                            50   0                                          100    8 × 10.sup.6                                                                       15    100(0-200)                                                                             0    0                                          100    .sup. 5 × 10.sup.6.spsp.d                                                           6    1600    800  200                                anti-IgE.sub.id                                                                        50     3.5 × 10.sup.6                                                                      4    100(0-200)                                                                             --   0                                  nRGG    100    5 × 10.sup.6                                                                       12    1600    --   400                                                                            (200)                              none    --     --        15    1600    --   400                                ______________________________________                                          .sup.a Normal spleen cells (10.sup.7) were cultured in miniMarbrook            chambers with antiT.sub.HF, antiIgE.sub.id, or nRGG for 4 days at              37° in 10% CO.sub.2 and 7% O.sub.2.                                     .sup.b Upon completion of culture, cells are washed three times in PBS, p      7.2, counted, and the indicated number of viable cells were injected           intravenously into recipients primed 20 days earlier with 10 μg of WST      adsorbed on 1 mg of alum. Recip ients were given a secondary antigen           challenge with the same dose of WST within 24 hours after cell transfer.       .sup.c IgE measured by PCA in SpragueDawley rats in duplicate. Mean            response is reported and the range of response where observed is indicate      by parenthesis.                                                                .sup.d Cells cultured with 100 μg of antiTH.sub.F and treated with          antithy 1 sera and complement prior to the cells being injected                intravenously into recipients.                                           

Previous studies comparing the effectiveness of intact anti-T_(HF) and the F(ab)₂ fragment of anti-T_(HF) antibody to induce T_(S) cells in vivo showed that the F(ab)₂ fragment of anti-T_(HF) was unable to induce T_(S) cells. Since the in vivo persistance of the intact and F(ab)₂ fragment of anti-T_(HF) differs significantly, the ability of the F(ab)₂ fragment to induce T_(S) cells in the in vitro culture system was reevaluated. Table 10 shows that the F(ab)₂ fragment of anti-T_(HF) cultured 4 days with normal spleen cells fails to induce T_(S) cells. In addition, cultures of B-cell-depleted (cells nonadherent to anti-mouse IgM-coated petri dishes) spleen cells with anti-T_(HF) antibody did not result in the induction of T_(S) cells (Table 10).

                  TABLE 10                                                         ______________________________________                                         The requirement of F.sub.C.sup.+  cells for the in vitro induction             of suppressor T cells by anti-idiotypic antibody.                                         Source of          No. of                                                                               IgE response.sup.c                         Dose       normal   No. of cells                                                                             recip-                                                                               Day                                        Treatment.sup.a                                                                        (μg)                                                                               cells    transferred.sup.b                                                                      ients 7    14                                  ______________________________________                                         nRGG    100    spleen   5 × 10.sup.6                                                                     4     1600 600                                 anti-T.sub.HF                                                                          50     spleen   5 × 10.sup.6                                                                     8      50   0                                  anti-T.sub.HF                                                                          60     spleen   7 × 10.sup.6                                                                     4     1600 400                                 F(ab).sub.2 d                                                                  anti-T.sub.HF                                                                          50     B cell   7 × 10.sup.6                                                                     6     1250 600                                                depleted.sup.e                                                  ______________________________________                                          .sup.a Normal spleen cells (10.sup.7) were cultured in miniMarbrook            chambers with the indicated doses of nRGG, antiT.sub.HF, or the                F(ab).sub.2 fragment of antiT.sub.HF for 4 days at 37° in 10%           CO.sub.2 and 7% O.sub.2 .                                                      .sup.b Upon completion of culture, cells were washed three times in PBS,       pH 7.2, counted, and the indicated number of viable cells were injected        intravenously into recipients primed 20 days earlier with 10 μg of WST      adsorbed on 1 mg of alum. Reci pients were given a secondary antigen           challenge with the same dose of WST within 24 hours after cell transfer.       .sup.c IgE measured by PCA in SpragueDawley rats in duplicate and the mea      response is reported.                                                          .sup.d Pepsin digest of antiT.sub.HF antibody isolated by passage over a       Sepharoseprotein A adsorbent.                                                  .sup.e Spleen cells nonadherent to sheep antimouse IgM coated petri            dishes.                                                                  

In an effort to determine the nature of T_(S) cells induced by anti-Id, normal spleen cells were cultured with 100 μg of anti-T_(HF) antibody or 100 μg of nRGG. Upon determination of the cultures, the cells were washed three times with PBS, pH 7.2, and the cells frozen (at -20° C.) and thawed (at 37° C.) three times. The cell-free supernatant from pooled lysates was obtained by centrifugation at 20,000 rpm in a Beckman ultracentrifuge, and stored at -20° C. until passed over Sepharaose-AgD and Sepharose-T_(SF) adsorbents. Table 11 shows that both T_(SF).sbsb.1 and T_(SF).sbsb.2 factor were extracted from spleen cells cultured with anti-Id. On the other hand, neither T_(SF).sbsb.1 nor T_(SF).sbsb.2 was extracted from spleen cells cultured with nRGG. The level of T_(SF).sbsb.1 and T_(SF).sbsb.2 factor present in each cell-free supernatant was determined by an ELISA of material eluted from Sepharose-AgD and Sepharose-T_(SF) adsorbents.

                  TABLE 11                                                         ______________________________________                                         Soluble factors extracted from normal spleen cells                             cultured with anti-T.sub.HF antibody.                                                       Factors eluted from                                               Cells     Dose     Sepharose-AgD                                                                              Sepharose-T.sub.SF                              cultured with                                                                            (μg)  T.sub.SF.sbsb.2 /10.sup.8 cells                                                            - ald/10.sup.8 cells                            ______________________________________                                         anti-T.sub.HF                                                                            100      0.44 mg     0.14 mg                                         nRGG      100      0           0                                               ______________________________________                                    

EXAMPLE 4 In vitro induction of suppressor T cells with antigen B-specific T suppressor factor

                  TABLE 12                                                         ______________________________________                                                                           IgE response.sup.c                           Dose         No. of cells                                                                             No. of     Day                                          Treatment.sup.a                                                                        (μg)  transferred.sup.b                                                                        recipients                                                                              7    14                                    ______________________________________                                         nRGG    50       8 × 10.sup.6                                                                       4        1600 600                                   T.sub.SF.sup.d                                                                          5       5 × 10.sup.6                                                                       6        1000 400                                           25       5 × 10.sup.6                                                                       8         700 100                                           50       5 × 10.sup.6                                                                       6         100  0                                            100      5 × 10.sup.6                                                                       4         100  0                                            100      .sup. 7 × 10.sup.6.spsp.e                                                          4        1600 400                                   T.sub.SF.sbsb.2.sup.f                                                                  50       5 × 10.sup.6                                                                       4          0   0                                    ______________________________________                                          .sup.a Normal spleen cells (10.sup.7) were cultured in miniMarbrook            chambers with nRGG or T.sub.SF for 4 days at 37° in 10% CO.sub.2        and 7% O.sub.2.                                                                .sup.b Upon completion of culture, cells are washed three times in FBS, p      7.2, counted, and the indicated number of viable cells were injected           intravenously into recipients primed 20 days earlier with 10 g of WST          adsorbed on 1 mg of alum. Recipient s were given a secondary antigen           challenge with the same dose of WST within 24 hours after cell transfer.       .sup.c IgE measured by PCA in SpragueDawley rats in duplicate and the mea      response is reported.                                                          .sup.d Represents antigen Bspecific T suppressor factor (T.sub.SF)             isolated by affinity chromatography on Sepharoseantigen D.                     .sup.e Cells cultured with 100 μg of T.sub.SF and treated with antithy      1 sera and complement prior to the cells being injected intravenously int      recipients.                                                                    .sup.f Antigen B specific Tsuppressor factor (T.sub.SF.sbsb.2) isolated b      affinity chromography on SepharoseT.sub.SF.                              

EXAMPLE 5 Induction of T_(S) cells with Ox-AgB and preparation of T_(SF).

Timothy pollen extract (WST), purified AgB, Ox-AgB, antigen D₁ (AgD₁), and keyhole limpet (Megathura crenulata) hemocyanin (KLH) were prepared as described [Malley, A., Baecher, L., Begley, D., Dev. Biol. Standard, 29: 29 (1975); Malley, A., Begley, D., and Forsham, A., Immunochemistry (in press, 1979); Campbell, D. H., Garvey, J. S., Cremer, N. E., Sussdorf, D. H., Methods in Immunology, p. 69 (Benjamin, New York, 1963)]. The protein content of the samples was determined by the method of Lowry [Markwell, M. A. K., Haas, S. M., Beiber, L. L., and Tolbert, N. E., Analyt. Biochem. 87: 206 (1978)]. The carbohydrate content of the suppressor factor was described by the orcinol method [Ashwell, G., Methods in Enzymology, VIII: 94 (1966)].

LAF₁ mice (Jackson Laboratory, Bar Harbor, Me.) were immunized with 10 μg of WST adsorbed to 1 mg of alum on days 0 and 21; 7 days later spleen cells were removed to obtain immune spleen cells.

LAF₁ mice received 100-200 μg of Ox-AgB in complete Freund's adjuvant (CFA) in each of two injections at a 14-day interval. Seven days after the second priming, spleen cells were removed; these either were used in adoptive transfer to measure T_(S) cells or were placed in culture with isolubilized antigen (Sepharose-AgD₁) to obtain secreted T_(S) factor (T_(SF))

For the kinetics studies, animals received 200 μg of Ox-AgB in CFA in each of two injections at a 14-day interval. Spleen cells were removed 3, 7, 10, 14 and 21 days after the second treatment with Ox-AgB. Immunizations were scheduled so that spleen cell removal for all groups fell on a single day.

Immune spleen cells were obtained from mice immunized with 10 μg of protein of WST. A single-cell suspension was prepared as described by Fairchild, S. S.; Malley, A., J. Immun. 97: 559 (1966), and 1.5-2×10⁸ cells were applied to nylon wool columns to provide immune T cells [Julius, M. H.; Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3: 645 (1973)]. Immune B cells were prepared by treatment of immune spleen cells (10⁷ cells/ml) with rabbit anti-thy 1.2 sera (Accurate Chemical Co., Hicksville, N.W.) and guinea pig complement (Colorado Serum Co., Denver, Colo.), at a final dilution of 1:10.

Syngeneic X-irradiated (600 rad) mice received i.v. injections of 2.5×10⁴ immune B cells and 4×10⁴ immune T cells. Within 4 hours all animals were challenged i.p. with 10 μg of WST in alum; blood samples were collected 7, 10, and 14 days later. The maximum IgE response was obtained 7 days after antigen challenge and reached an IgE titer of 800.

When suppressor cells were tested, 5×10⁷ cells from mice immunized with Ox-AgB in CFA or 5×10⁷ normal spleen cells were added and injected i.v. with the immune T and B cells. When suppressor factor was being assayed, a cell-free supernatant (CFS) equivalent to 5×10⁷ cells was added to the mixture of immune T and B cells and was injected i.v. into syngeneic X-irradiated recipients. The IgE titers (reciprocal of the lowest dilution giving a positive reaction) were measured by passive cutaneous anaphylaxis (PCA) as described by Ovary et al, J. Immun. 97: 559 (1966); and Fairchild et al, J. Immun. 115: 446 (1975); IgG responses were evaluated by a modified version of the radioallergosorbent test [Malley et al, Int. Archs. Allergy Appl. Immun. 62: 276 (1980)] with AgB-conjugated Whatman 3-mm paper disks.

The Sepharose-AgD₁, adsorbent was prepared in the following manner. Sepharose-AH-4B (Pharmacia Fine Chemicals, Piscataway, N.J.) was washed with 0.5 M NaCl, water, and 1.0 M NaH-CO₃, pH 9. 50 mg of AgD₁ were added to 10 ml of 0.5 M NaHCO₃ (pH 9), and the pH of the AgD₁ solution was adjusted to pH 10.5 just prior to addition of cyanogen bromide in dimethylformamide. 1.5 g of CNBr dissolved in 2 ml of dimethylformamide were added dropwise to the magnetically stirred AgD₁ solution; the pH was maintained between 10.0 and 10.7 by the addition of 5 N NaOH. When the pH was stable (within 10-15 min.), this solution was added to the Sepharose-AH slurry. Additional (10 ml) 0.1 M NaHCO₃ buffer (pH 9) was used to wash out the vessel, and the wash was added to the mixture and stirred at 4° C. overnight (20 hours).

The AgD₁ -conjugated Sepharose-AH was then filtered on a coarse sintered glass filter, and the matrix was washed with 300 ml of 0.1 M NaHCO₃ buffer, pH 9. The Sepharose-AH-AgD₁ was transferred to a beaker and stirred for 3 hours at room temperature with 1.0 M ethanolamine in 0.1 M NaHCO₃, pH 9. The matrix was washed on a sintered glass filter with the following solutions (250 ml each): 0.1 M NaHCO₃ -0.5 M NaCl, pH 10; distilled water; 0.1 M NaAc-0.5 M NaCl, pH 4; distilled water; 2 M urea-0.5 M NaCl; distilled water; and 0.01 M cacodylate buffer-0.155 M NaCl, pH 7.0. The Sepharose-AgD₁ adsorbent was stored at 4° C. in 0.01 M cacodylate-saline buffer (CSB), pH 7.0.

Spleen cells from mice immunized with Ox-AgB in CFA were used to obtain secreted soluble T_(SF). Each culture contained 1 ml of cells (5×10⁴ cells/ml) in RPMI 1640 supplemented with 5% heat-inactivated fetal calf serum. Antigenic stimulation of these primed cells was achieved by addition of one of the following: 100 μg of soluble AgB; 100 μg of a 1:5 suspension of Sepharose-AgB; or 100 μl of a 1:5 suspension of a Sepharose-AgD₁ conjugate in RPMI 1640. Cultures were incubated at 37° C. for 24, 48, or 72 hours in 5% CO₂ and 95% air. Upon completion of these cultures, the cells were centrifuged and the CFSs were collected. Each CFS was tested by adoptive transfer for the presence of T_(SF). Recipients were given CFS equivalent to 5×10⁷ cells and immune T and B cells. Blood samples were obtained 7 and 14 days later, and IgE antibody titers were determined by PCA.

Immune spleen cells from mice primed twice with 100 μg of Ox-AgB in CFA (as described above) were sonicated in a Biosonik III apparatus (Bronwill Sci., Rochester, N.Y.) at 42 W/cm². The cells in 2 ml of media were placed in a 12×100 mm centrifuge tube and sonicated for 1.5 min. in an ice bath. The sonicate was centrifuged at 20,000 rpm for 30 min. at 4° C., and the CFS was collected and assayed for suppressor factors.

Spleen cells from mice primed with Ox-AgB in CFA were used to prepare both sonicate-derived and secreted soluble suppressor factors. Sonicate-derived (T_(SF-E)) or secreted T suppressor factor (T_(SF-S)) was passed over a Sepharose-AgD₁ adsorbent (10 ml of packed resin) equilibrated in CSB, pH 7. The nonadherent protein fraction was washed through the adsorbent with CSB, and the adsorbent was washed with 5-10 column volumes. The adherent fraction was eluted with 15 ml of 3 M KSCN in CSB, and the adsorbent was washed with an additional 100 ml of CSB. The first 35-50 ml of eluted material was collected, dialyzed, concentrated by negative pressure against CSB. Both nonadherent and adherent fractions were tested for the presence of suppressor factor by adoptive transfer.

The suppresive capacity of spleen cells from mice primed with Ox-AgB in CFA is shown in Table 12. In previous studies [Malley et al, Int. Archs. Allergy Appl. Immun. 62:276 (1980)], adoptive transfer recipients receiving 4×10⁶ WST-immune T cells and 2.5×10⁶ WST-immune B cells and challenged with 10 μg of WST in alum produced a maximum IgE response (PCA titer of 800) 7 days later. The addition of 5×10⁷ spleen cells from mice primed with CFA only to this mixture of WST-immune T and B cells did not reduce the secondary IgE response obtained in adoptive transfer recipients. In contrast, spleen cells from mice primed with Ox-AgB in CFA added to the mixture of immune cells resulted in dose-dependent suppression of the secondary IgE response. Treatment of the Ox-AgB-primed spleen cells with anti-thy 1.2 sera and guinea pig complement resulted in a complete loss of suppressive activity in the remaining cells. Antigenic specificity of the T_(S) cells for AgB was demonstrated by the failure of Ox-AgB-primed spleen cells to influence a secondary IgE response to ovalbumin in synageneic X-irradiated recipients.

                  TABLE 12                                                         ______________________________________                                         Suppression of the anti-antigen-B IgE response                                 by T suppressor cells                                                                             Ratio                                                                          immune                                                      Source   Number    cells.sup.1                                                                               Irradiated                                                                             PCA                                      of cells of cells  T:B        recipients.sup.2                                                                       titer.sup.3                              ______________________________________                                         Ox--AgB  5 × 10.sup.7                                                                       1.6:1      10       0                                       CFA.sup.4                                                                      Ox--AgB  2.5 × 10.sup.7                                                                     1.6:1      10      200                                      CFA                                                                            Ox--AgB  10.sup.7  1.6:1      10      400                                      CFA                                                                            Ox--AgB  5 × 10.sup.6                                                                       1.6:1      10      800                                      CFA                                                                            Ox--AgB  5 × 10.sup.7                                                                       1.6:1       3      800                                      CFA.sup.5                                                                      FCA only.sup.6                                                                          5 × 10.sup.7                                                                       1.6:1       3      800                                      ______________________________________                                          .sup.1 Mice were primed twice with 10 μg of WST adsorbed on 1 mg of         alum at 21day intervals. Spleen cells were prepared 7 days later. Immune       cells represent nylon woolnonadherent cells (1.5 × 10.sup.8 cells        applied to column); immune B cells represent spleen cells treated with         antithy 1.2(1:10) and guinea pig complement (1:10).                            .sup.2 All cells were injected i.v. into Xirradiated (600 rad) syngeneic       mice, and were challenged with 10 μ g of WST adsorbed on 1 mg of alum       within 4 hours after injection of the cells.                                   .sup.3 Passive cutaneous anaphylaxis was accomplished in duplicate; the        indicated titer represents the reciprocal of the lowest dilution with a 5      × 5 mm reaction.                                                         .sup.4 Mice were primed twice with 100 μg of Ox--AgB in CFA at 14day        intervals, and the spleen cells were prepared 7 days later.                    .sup.5 Spleen cells from mice primed with Ox--AgB in CFA and treated with      antithy 1.2 sera and guinea pig complement.                                    .sup.6 Spleen cells from mice primed with CFA only.                      

The kinetics of T_(S) cell induction in mice primed with Ox-AgB in CFA at 14-day intervals is shown in Table 13. Although some T_(S) cells were evident within 3 three days after the last injection of Ox-AgB, the maximum level of induction occurred between days 7 and 14 and T_(S) cells were absent in the spleen 21 days after Ox-AgB injection. In subsequent studies designed to either evaluate the activity of such cells or isolate T_(SF), spleen cells from Ox-AgB-primed mice were obtained 7-10 days after the last injection of Ox-AgB.

                  TABLE 13                                                         ______________________________________                                         Kinetics of the induction of T suppressor cells                                with antigen B modified by photooxidation.                                     Days after Ratio of cells                                                      last injection                                                                            used in adoptive                                                                              Number of PCA                                        of Ox--AgB.sup.1                                                                          transfer.sup.2 recipients                                                                               titer.sup.3                                ______________________________________                                          3         1.6:1          6         200                                         7         1.6:1          6          50                                        10         1.6:1          6         100                                        14         1.6:1          6          0                                         21         1.6:1          6         800                                        None.sup.4 1.6:1          6         800                                        ______________________________________                                          .sup.1 Mice were primed twice with 200 μg of Ox--AgB in CFA at 14day        intervals; spleen cells were collected at the indicated interval after th      last injection of Ox--AgB.                                                     .sup.2 Mice were primed with 10 μg of WST adsorbed on 1 mg of alum at       21day intervals. Spleen cells were prepared 7 days later. Immune T cells       represent nylon wool nonadherent cells; immune B cells represent spleen        cells treated with antithy 1 .2 (1:10) and guinea pig complement (1:10).       Immune T and B cells were mixed in a ratio of 1.6:1 (4 × 10.sup.6        T/2.5 × 10.sup.6 B cells) with 5 × 10.sup. 7 Ox--AgBprimed         cells and injected i.v. into Xirradiated (600 rad) syneneic m ice. All         recipients were challenged with 10 μg of WST adsorbed on 1 mg of alum       within 4 hours after injection of the cells.                                   .sup.3 Passive cutaneous anaphylaxis was accomplished in duplicate in          SpragueDawley rats. The indicated titer represents the reciprocal of the       lowest dilution with a 5 × 5 mm reaction.                                .sup.4 Spleen cells from mice primed with CFA only (cells were collected       days later).                                                             

The conditions for the secretion of T_(SF-S) (S-secreted) from Ox-AgB-specific cells are shown in Table 14.

                                      TABLE 14                                     __________________________________________________________________________     Tissue culture conditions for the secretion                                    of soluble T suppressor factor.                                                                      Ratio of                                                                       cells used                                                       Culture conditions.sup.1                                                                     in adoptive                                                                          Number of                                                                            PCA                                          Source of cells                                                                        antigen   time, h                                                                            transfer.sup.2                                                                       recipients                                                                           titer.sup.3                                  __________________________________________________________________________     Ox--AgB/CFA                                                                            sonicated.sup.4                                                                          --  1.6:1 6     200                                          Ox--AgB/CFA                                                                            AgB       24  1.6:1 6     800                                                  Sepharose-Ox--AgB                                                                        24  1.6:1 6     800                                                  Sepharose-AgD.sub.1                                                                      24  1.6:1 6     800                                          Ox--AgB/CFA                                                                            AgB       72  1.6:1 6     800                                                  Sepharose-Ox--AgB                                                                        72  1.6:1 6     100                                                  Sepharose-AgD.sub.1                                                                      72  1.6:1 6     100                                          KLH/CFA.sup.5                                                                          Sepharose-Ox--AgB                                                                        72  1.6:1 6     800                                                  Sepharose-AgD.sub.1                                                                      72  1.6:1 6     800                                          __________________________________________________________________________      .sup.1 Upon completion of tissue culture, the cells were removed by            centrifugation and the cellfree supernatant (CFS) collected. The amount o      CFS mixed with immune T and B cells was based upon the number of cells         used to obtain the CFS (5 × 1 0.sup.7 cell equivalents).                 .sup.2 Mice were primed with 10 μg of WST adsorbed on 1 mg of alum at       21day intervals and spleen cells were prepared 7 days later. Immune T          cells represent nylon wool nonadherent cells; immune B cells represent         spleen cells treated with antithy  1.2 sera (1:10) and guinea pig              complement (1:10). Immune T and B cells (4 × 10.sup.6 T. 2.5 .times      10.sup.6 B) were mixed with 5 × 10.sup.7 cell equivalents obtained       from Ox--AgBprimed cells either sonicated or cultured for 24-72 h ours         with antigen. These mixtures were injected i.v. into Xirradiated (600 rad      syngeneic recipients, and they were challenged with antigen within 4           hours.                                                                         .sup.3 Passive cutaneous anaphylaxis was accomplished in duplicate; the        indicated titer represents the reciprocal of the lowest dilution with a 5      × 5 mm reaction.                                                         .sup.4 Cells primed with 100 g of Ox--AgB in CFA were sonicated at 42          W/cm.sup.2 in a 12 × 100 mm tube for 1.5 min. on ice and centrifuge      at 20,000 rpm for 30 min. at 4° C. The sonicate was collected and       assayed for extracted suppress or factor.                                      .sup.5 Spleen cells from mice primed with KLH in CFA were cultured for 72      hours with insolubilized antigen (Ox--AgB and AgD.sub.1) and the CFS           collected.                                                               

Extraction of T_(SF-E) (E-extracted) by sonication of T_(S) cells results in the recovery of suppressor factor. The CFS (equivalent to 5×10⁷ cells) containing T_(SF-E) resulted in a 75% suppression of the AgB-specific IgE response. Attempts to induce the secretion of T_(SF-S) from Ox-AgB-specific T_(S) cells with soluble AgB or insolubilized Ox-AgB or AgD₁ during a 24-hour culture period were unsuccessful. However, Ox-AgB-specific T_(S) cells cultured for 72 hours with insolubilized antigen (Ox-AgB or AgD₁) secreted a significant amount (88% suppression) of T_(SF-S) into the culture supernatant. This factor was not secreted from Ox-AgB-primed T_(S) cells cultured with soluble antigen for 72 hours.

Partial purification of T_(SF) was achieved by affinity chromatography on a Sepharose-AH-Ox-AgB or Sepharose-AH-AgD₁ adsorbent equilibrated in 0.01 M CSB, pH 7. Material not adhering to the Sepharose-AgD₁ adsorbent did not contain any T_(SF) activity, but material eluted from the adsorbent with 3 M KSCN contained significant levels of this activity (Table 15). Analysis of T_(SF-E) and T_(SF-S) for protein and carbohydrate composition indicated that both partially purified factors contained about 5% carbohydrate and 95% protein.

                                      TABLE 15                                     __________________________________________________________________________     Partial purification of antigen B-specific                                     T suppressor factor by affinity chromatography.                                                        Ratio of                                                                       cells used                                             Source           Cell   in adoptive                                                                          Number of                                                                            PCA                                        spleen cells                                                                           Treatment                                                                               equivalents.sup.1                                                                     transfer.sup.2                                                                       recipients                                                                           titer.sup.3                                __________________________________________________________________________     CFA only                                                                               sonicate 5 × 10.sup.7                                                                    1.6:1 6     800                                        Ox--AgB/CFA                                                                            sonicate 5 × 10.sup.7                                                                    1.6:1 6     100                                        Ox--AgB/CFA                                                                            sonicate.sup.4                                                                          5 × 10.sup.7                                                                    1.6:1 6     800                                                Sepharose-AgD.sub.1                                                            nonadherent                                                            Ox--AgB/CFA                                                                            sonicate.sup.5                                                                          1 × 10.sup.7                                                                    1.6:1 6      50                                                Sepharose-AgD.sub.1                                                            adherent                                                               Ox--AgB/CFA                                                                            CFS(secreted).sup.6                                                                     5 × 10.sup.7                                                                    1.6:1 6     100                                        Ox--AgB/CFA                                                                            CFS(secreted).sup.7                                                                     5 × 10.sup.7                                                                    1.6:1 6     800                                                Sepharose AgD.sub.1                                                            nonadherent                                                            Ox--AgB/CFA                                                                            CFS(secreted).sup.8                                                                     2.5 × 10.sup.6                                                                  1.6:1 6      0                                                 Sepharose AgD.sub.1                                                            bound                                                                  __________________________________________________________________________      .sup.1 Cell equivalents represent the CFS obtained from a given number of      spleen cells treated by sonication or after tissue culture for 72 hours        (secreted).                                                                    .sup.2 Mice were primed with 10 μg of WST adsorbed on 1 mg of alum at       21day intervals and spleen cells were prepared 7 days later. Immune T          cells represent nylon wool nonadherent cells; immune B cells represent         spleen cells treated with antithy  1.2 sera (1:10) and guinea pig              complement (1:10). Immune T and B cells (4 × 10.sup.6 T/s. 5 .times      10.sup.6 B) were mixed with the indicated amounts of material derived fro      Ox--AgBprimed spleen cells. These mixtures were injected i.v. int o            Xirradiated (600 rad) syngeneic recipients, and they were challenged with      antigen within 4 hours.                                                        .sup.3 Passive cutaneous anaphylaxis was accomplished in duplicate in          SpragueDawley rats. The indicated titer represents the reciprocal of the       lowest dilution with a 5 × 5 mm reaction.                                .sup.4 Sonicate is from spleen cells of mice immunized with Ox--AgB/CFA        which has been partially purified on a SepharoseAH--AgB.sub.1 adsorbent.       Nonadherent fraction was used.                                                 .sup.5 Sonicate is from spleen cells of mice immunized with Ox--AgB/CFA        which has been partially purified on a SepharoseAH--AgD.sub.1 adsorbent.       Adherent fraction, eluted with 3 M KSCN, was used.                              .sup.6 CFS was obtained from spleen cells of mice primed with Ox--AgB in      CFA and cultured for 72 hours with insolubilized antigen.                      .sup.7 CFS was obtained as in `6` and partially purified on a                  SepharoseAH--AgD.sub.1 adsorbent. Nonadherent fraction was used.               .sup.8 CFS was obtained as in `6` and partially purified on a                  SepharoseAH--AgD.sub.1 adsorbent. Adherent fraction, eluted with 3 M KSCN      was used.                                                                 

I claim:
 1. A method of preparing an anti-idiotypic antibody directed against (1) Timothy grass antigen B- and (2) cross reactive grass antigen-specific allergic antibody, IgE, said anti-idiotypic antibody, anti-IgE_(id), being specifically reactive with the idiotypic determinants on said IgE and (1) antigen B- and (2) cross reactive grass antigen-specific T and B cells comprising:(1) immunizing a first animal species with a source of antigen B, (2) collecting sera from said immunized first animal species, (3) separating IgE from said sera, (4) immunizing a second animal species with said IgE, (5) collecting sera from said immunized second animal species, and (6) separating said anti-IgE_(id) antibody from said sera.
 2. The method of claim 1 wherein said second animal species is different from said first animal species.
 3. The method of claim 1 wherein said first animal species is mouse and said second animal species is rabbit.
 4. The method of claim 1 wherein said step (1) includes the step of hyperimmunizing said first animal species with a source of antigen B prior to the collection
 5. The antibody anti-IgE_(id).
 6. A method of preparing an anti-idiotypic antibody directed against (1) Timothy grass antigen B- and (2) cross reactive grass antigen-specific allergenic T-helper factor, T_(HF), said anti-idiotypic antibody, anti-T_(HF), being specifically reactive with the idiotypic determinants on said T_(HF) and (1) antigen B- and (2) cross reactive grass antigen-specific T and B cells comprising:(1) immunizing a first animal species with photooxidized antigen B, (2) collecting T_(H) - cells from said immunized first animal species, (3) culturing said T_(H) -cells with a source of Timothy grass antigen B or cross reactive grass antigen, (4) separating T-helper factor, T_(HF), from said culture, (5) immunizing a second animal species with said T-helper factor, T_(HF), (6) collecting sera from said immunized second animal species, and (7) separating said anti-T_(HF) antibody therefrom.
 7. The method of claim 6 wherein said second animal species is different from said first animal species.
 8. The method of claim 6 wherein said first animal species is mouse and said second animal species is rabbit.
 9. The method of claim 6 wherein said T_(H) -cells are collected from the spleen of said first animal species.
 10. The method of claim 6 wherein said first animal species is immunized with photooxidized antigen B adsorbed on alum.
 11. The method of claim 6 wherein said culture medium containing T_(HF) is centrifuged and T_(HF) isolated from the cell-free supernatant by affinity chromatography.
 12. The method of claim 6 wherein said anti-T_(HF) is isolated from sera by affinity chromatography.
 13. The antibody, anti-T_(HF).
 14. A method of producing (1) Timothy grass antigen B- and (2) cross reactive grass antigen-specific T suppressor cells, T_(S), comprising:(1) culturing the antibody of claim 5 or 13 with normal cells of an animal to induce the formation of T_(S) cells in said culture and (2) separating said T_(S) from said culture.
 15. The method of claim 14 wherein said normal cells comprise spleen cells.
 16. The method of claim 14 wherein said normal cells comprise human peripheral blood lymphocytes.
 17. A method of producing Timothy grass antigen B- and crossreactive grass antigen-specific T suppressor cells, T_(S), comprising:1. immunizing an animal species with anti-IgE_(id) and,
 2. collecting T_(s) cells from said animal species.
 18. The method of claim 17 wherein said T_(S) - cells are derived from the spleen of said immunized animal species.
 19. A method of producing (1) Timothy grass antigen B- and (2) cross reactive grass-antigen specific suppressor T-cell factor, T_(SF), by repeatedly freezing and thawing Ts cells and separating said Ts cells from T_(SF).
 20. The method of claim 19 wherein said T_(SF) is isolated by affinity chromatography.
 21. A method for producing (1) Timothy grass antigen B- and (2) cross reactive grass antigen-specific suppression T-cell factor, T_(SF), comprising:(1) culturing T suppressor cells, T_(S), with antigen-IgE_(id), and (2) separating T_(SF) from said cell containing culture.
 22. The method of claim 21 wherein said T_(SF) containing medium is centrifuged to obtain a cell-free supernatant and T_(SF) is recovered from said supernatant by affinity chromatography.
 23. A method of fractionating T_(SF) into separate factors, T_(SF).sbsb.1 and T_(SF).sbsb.2, comprising the steps of: (1) subjecting T_(SF) to affinity chromatography on an adsorbent comprising Timothy grass antigen D₁ on an inert carrier substrate; (2) collecting the non-adsorbing fraction; (3) eluting T_(SF).sbsb.1 adsorbed on said adsorbent and separating T_(SF).sbsb.1 from the eluant; (4) subjecting the non-adherent fraction to affinity chromotagraphy on an adsorbent comprising T_(SF).sbsb.1 on an inert carrier substrate, eluting the T_(SF).sbsb.2 therefrom and separating T_(SF).sbsb.2 from the eluant.
 24. The protein fraction T_(SF).sbsb.2.
 25. A method of producing Timothy grass antigen B-and cross reactive grass antigen-specific T suppressor cells, T_(S), comprising:(1) culturing T_(SF).sbsb.1 or T_(SF).sbsb.2 with normal cells of an animal species, and (2) separating T_(S) from said normal cell containing culture.
 26. A pharmaceutical composition in unit dosage form for suppressing the allergic response of an animal sensitive to Timothy grass antigen B or a cross reactive grass antigen comprising an anti-allergic response effective amount of the product of claims 5, 13, or 24 and a pharmaceutically acceptable carrier therefor.
 27. A method for suppressing the allergic response of an animal sensitive to Timothy grass antigen B or a cross reactive grass antigen comprising administering thereto an anti-allergic response effective amount of the product of claims 5, 13, or
 24. 28. A pharmaceutical composition in unit dosage form for suppressing the allergic response of an animal sensitive to Timothy grass antigen B or a cross reactive grass antigen comprising an anti-allergic response effective amount of the product when made by the process of claims 19 or
 23. 29. A method for suppressing the allergic response of an animal sensitive to Timothy grass antigen B or a cross reactive grass antigen comprising administering thereto an anti-allergic response effective amount of the product when made by the process of claims 19 or
 23. 